Steroid hormones, including glucocorticoids (GCs), play an important role in the regulation of the immune system (Chrousos, G. P., N. Engl. J. Med. 332, 1351-1362 (1995)). Endogenous glucocorticoid synthesis is controlled by the hypothalamic-pituitary-adrenal axis (Chrousos, G. P., N. Engl. J. Med. 332, 1351-1362 (1995); Rhen, T., & Cidlowski, J. A., N. Engl. J. Med. 353, 1711-1723 (2005)) and is regulated by the transcriptional control of steroidogenic enzymes of the cytochrome P450 gene family (Mueller, M., et al. J. Exp. Med. 203, 2057-2062 (2006)). Corticosteroids have been used in treating allergic diseases due to their anti-inflammatory activity (Barnes, P J. Br. J. Pharmacol. 163:29-43 (2011)), but, somewhat paradoxically, increasing evidence indicates that corticosteroids may also enhance disease pathogenesis by activating and enhancing growth of CD4 T cells and inhibiting Th1 cytokine production (Cima, I., Fuhrer, A., & Brunner, T. Immunol. Lett. 106, 99-102 (2006)). Glucocorticoids amplified immune responses in steroid-insensitive CD8+ T cells (Ohnishi, H., et al. J. Allergy Clin. Immunol. 121, 864-871 (2008)). As well, the corticosteroids themselves may induce Th2 cytokine production while simultaneously suppressing the production of Th1 cytokines (Koya, T. et al. J. Immunol. 179, 2787-2796 (2007)).
The inhibitory role of GCs on immune cells is well characterized (De Bosscher, K., et al. Endocr. Rev. 24, 488-522 (2003); De Bosscher, K, & Haegeman, G. Mol. Endocrinol. 23, 281-291 (2009)). GCs reduce inflammation through inhibition of NF-κB and by inducing the expression of anti-inflammatory proteins including annexin 1 and MAPK phosphatase 1 (Chrousos, G. P. N. Engl. J. Med. 332, 1351-1362 (1995)). GCs and other synthetic derivatives have been used to treat a variety of diseases, including inflammatory diseases of the intestine and asthma (Barnes, P J. Br. J. Pharmacol. 163:29-43 (2011); Faubion, W. A. Jr., et al. Gastroenterology 121, 255-260 (2001)). Although the anti-inflammatory activity of GCs is well described, accumulating evidence suggests that GCs can also enhance immune cell activation, inducing gene transcription and promoting the pathogenesis of allergic diseases (Cima, I., et al. J. Exp. Med. 200, 1635-1646 (2004); Ohnishi, H., et al. J. Allergy Clin. Immunol. 121, 864-871 (2008)). Steroid hormones are mainly produced in the adrenal glands, but other tissues also produce GCs through the induction of steroidogenic enzymes (Chrousos, G. P. N. Engl. J. Med. 332, 1351-1362 (1995); Payne, A. H. Biol. Reprod. 42, 399-404 (1990)). The intestinal mucosa contains steroidogenic enzymes such as cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1) and synthesizes potent GCs which exhibit both an inhibitory and a co-stimulatory role on intestinal T cell activation (Cima, I., et al. J. Exp. Med. 200, 1635-1646 (2004)).
Cyp11a1 (also known as P450scc) is a key regulator of steroid biogenesis as the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone (Pazirandeh, A., et al. FASEB J. 13, 893-901 (1999)). Induction of the Cyp11a1 promoter by epidermal growth factor involves a ras/MEK1/AP-1-dependent pathway (Croft, M. et al. J. Exp. Med. 180, 1715-1728 (1994)). Cyp11a1 is expressed primarily in the cortex of the adrenal gland, but testis, ovary, placenta, thymus, and intestine also express Cyp11a1 (Cima, I., et al. J. Exp. Med. 200, 1635-1646 (2004); Pazirandeh, A., et al. FASEB J. 13, 893-901 (1999)). Activation of Cyp11a1 results in a spectrum of steroid hormones, including glucocorticoids that are known to play a role in T cell function (Mosmann, T. R., and Coffman, R. L. Annu. Rev. Immunol. 7, 145-173 (1989); Seder, R. A. et al. J. Immunol. 148, 1652-1656 (1992)). Several of the gonadal steroids have been shown to have important immune effects on T cells that express their cognate receptors. T cells express receptors for androgen and estrogen and receptor activation can impact cytokine gene transcription. These studies have related gender bias to differences in the response of CD4, CD8, and T regulatory cells (De Bosscher, K., et al. Endocr. Rev. 24, 488-522 (2003); De Bosscher, K, & Haegeman, Mol. Endocrinol. 23, 281-291 (2009)). T cells also express many of the steroid metabolic enzymes (De Bosscher, K., et al. Endocr. Rev. 24, 488-522 (2003)). Depletion of Cyp11a1 in mice or rabbits results in steroid deficiency, female external genitalia, and death (Shih, M. C., et al. Mol. Cell. Endocrinol. 336, 80-84 (2011); Pang, S., et al. Endocrinology 131, 181-186 (1992); Yang, X., et al. Endocrinology 132, 1977-1982 (1993)). In humans, mutations in the Cyp11a1 gene result in a steroid hormone deficiency, causing a rare and potentially fatal form of lipoid congenital adrenal hyperplasia (Kim, C. J., et al. J. Clin. Endocrinol. Metab. 93, 696-702 (2008); Al Kandari, H., et al. J. Clin. Endocrinol. Metab. 91, 2821-2826 (2006)). Patients with a heterozygous or homozygous mutation of Cyp11a1 exhibit adrenal insufficiency and sex reversal (Tajima, T., et al. J. Clin. Endocrinol. Metab. 86, 3820-3825 (2001); Parajes, S., et al. J. Clin. Endocrinol. Metab. 96, E1798-E1806 (2011)).
Transcription factors such as Steroidogenic Factor-1 (SF-1), Activator Protein 2 (AP-2), and several tissue-specific GATA family proteins enhance the transcription of Cyp11a1 through interactions with AP-1, specificity Protein-1 (SP-1) and AP-2 (National Asthma Education and Prevention Program (National Heart Lung and Blood Institute) Third Expert Panel on the Management of Asthma. National Center for Biotechnology Information (U.S.). Expert panel report 3 guidelines for the diagnosis and management of asthma. Bethesda, Md.: National Institutes of Health National Heart Lung and Blood Institute; 2007). In particular, the GATA protein family plays an important role in the regulation of Cyp11a1 expression (Barnes, P J. Br J. Pharmacol. 163:29-43 (2011)). GATA binding elements have been identified in the Cyp11a1 promoter and Cyp11a1 expression was decreased in GATA3-deficient mice (Wei, G, et al. Immunity 35, 299-311 (2011)). GATA4 significantly upregulated Cyp11a1 expression in granulosa cells (Sher, N., et al. Mol. Endocrinol. 21, 948-962 (2007)). These results identify important events in the transcriptional regulation of Cyp11a1 that directly affect steroid synthesis and release.
CD4 Th cells play a pivotal role in the induction and control of allergic inflammation, including food allergy (Islam, S. A., & Luster, A. D. Nature Med. 18, 705-715 (2012)). In a mouse model of food allergy, allergen-specific CD4 T cells were activated in the mesenteric lymph nodes and recruited to the small intestine, resulting in increased levels of Th2 cytokines in the inflamed small intestine (Knight, A. K., et al. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G1234-G1243 (2007)).
In humans, allergen-specific Th2 CD4 T cells are essential in the development and maintenance of both type I IgE-mediated and non-IgE-mediated food allergic responses. In patients with anaphylactic peanut allergy, increased numbers of peanut-specific IL-5- and IL-4-producing Th2 cells are found in peripheral blood (Prussin, C., et al. J. Allergy Clin. Immunol. 124, 1326-1332 (2009)). In addition, peanut-specific T cell lines from individuals with peanut anaphylaxis primarily produce Th2 cytokines (IL-4, IL-13) (DeLong, J. H., et al. J. Allergy Clin. Immunol. 127, 1211-1218 (2011)). Other food allergies were also characterized by increased levels of Th2 cytokines; in patients with milk-induced gastrointestinal diseases, milk-specific CD4 T cells derived from the duodenal mucosa produce high levels of Th2 cytokines, especially IL-13 (Beyer, K., et al. J. Allergy Clin. Immunol. 109, 707-713 (2002)).
Allergic asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic airway inflammation and airway hyperresponsiveness (AHR) (Kim, H. Y., et al. Nat. Immunol. 11, 577-584 (2011); Holgate, S. T. Nat. Med. 18, 673-683 (2012)). Numbers of CD8+IL-13+ T cells are increased in asthmatics (Gelfand, E. W. and Dakhama, A. J. Allergy Clin. Immunol. 117, 577-582 (2006)) and during the development of experimental asthma in mice (Hamelmann, E. et al. J. Exp. Med. 183, 1719-1729 (1996); Miyahara, N. et al. J. Immunol. 172, 2549-2558 (2004); Miyahara, N. et al. J. Immunol. 174, 4979-4984 (2005)). In an atopic environment rich in IL-4, these CD8+ T cells mediate asthmatic responses (Koya, T. et al. J. Immunol. 179, 2787-2796 (2007)). However, the mechanisms regulating the conversion of CD8+ effector T cells from IFN-γ to pathogenic IL-13-producing effector cells have not been defined.
Asthma has increased dramatically over the past 50 years and now affects 5-10% of the population in many developed countries (Kim, H. Y., et al. Nat. Immunol. 11, 577-584 (2011)). National and international guidelines recommend the use of inhaled corticosteroids as the first step in controlling airway inflammation and symptoms in persistent asthma (Holgate, S. T. Nat. Med. 18, 673-683 (2012); Gelfand, E. W. and Dakhama, A. J. Allergy Clin. Immunol. 117, 577-582 (2006)). However, it has been demonstrated that 45% of steroid-naive asthmatic patients do not respond to inhaled corticosteroids. Corticosteroid insensitivity has been adopted as a principal criterion for characterizing asthma severity (Hamelmann, E. et al. J. Exp. Med. 183, 1719-1729 (1996)). Increased numbers of CD8+ T cells, which are more resistant than CD4+ T cells to corticosteroids (Miyahara, N. et al. J. Immunol. 172, 2549-2558 (2004); Miyahara, N. et al. J. Immunol. 174, 4979-4984 (2005)), have been detected in steroid-insensitive asthmatics (Koya, T. et al. J. Immunol. 179, 2787-2796 (2007)) and have correlated with lower lung function (LaVoie, H. A. and King, S. R. Exp. Biol. Med. 234, 880-907 (2009)). The inventors and others also found that numbers of CD8+IL-13+ cells were increased in experimental asthma models in mice (Shih, M. C. et al. Mol. Endocrinol. 22, 915-923 (2008); National Asthma Education and Prevention Program (National Heart Lung and Blood Institute) Third Expert Panel on the Management of Asthma. National Center for Biotechnology Information (U.S.). Expert panel report 3 guidelines for the diagnosis and management of asthma. Bethesda, Md.: National Institutes of Health National Heart Lung and Blood Institute; 2007, Guidelines for the diagnosis and management of asthma. Bethesda, Md.: National Institutes of Health National Heart Lung and Blood Institute; 2007) as a result of their activation by IL-4-producing CD4+ T cells (Martin, R. J. et al. J. Allergy Clin. Immunol. 119, 73-80 (2007)). CD8+ T cells can be polarized to effector subsets with cytokine profiles similar to those found in CD4+ T cells (Li, L. B. et al. Blood 110, 1570-1577 (2007); Payne, A. H. Biol. Reprod. 42, 399-404 (1990); van Rensen, E. L. et al. Am. J. Respir. Crit. Care Med. 172, 837-841 (2005)). Both in vivo and in vitro, IL-4 is capable of triggering CD8+ T cell differentiation from a predominant IFN-γ-producing cell to one producing IL-13. However, the mechanisms underlying this conversion of CD8+ T cells is unknown.
Transcriptional profiling identified Cyp11a1 transcripts as one of the most highly up-regulated during the differentiation of CD8+ T lymphocytes to a Tc2 phenotype, that is, a CD8 T cell capable of IL-13 production. This upregulation of Cyp11a1 in CD8+ T cells is similar to the upregulation seen in CD4+ T cells in a peanut allergy model, suggesting that this enzyme is essential in CD4+ and CD8+ T cells for pro-allergic differentiation.
CD4+ T cell differentiation into Th2 cells with production of IL-4, IL-5, IL-9, and IL-13 has been shown to be critical for the development of altered airway responsiveness and eosinophilic airway inflammation in experimental models of asthma (Samy, T. S. et al. Endocrinology 142, 3519-3529 (2001); Pottratz, S. T. et al. J. Clin. Invest. 93, 944-950 (1994)). In addition to CD4+ T cells, CD8+ T cells can be polarized to effector subsets with cytokine profiles similar to those found in CD4+ T cells (Payne, A. H. Biol. Reprod. 42, 399-404 (1990); van Rensen, E. L. et al. Am. J. Respir. Crit. Care Med. 172, 837-841 (2005)). It has been previously demonstrated that there is an important role for type 2 (Tc2) CD8+ T cells in the development of experimental asthma (Slominski, A. et al. FEBS J. 273, 2891-2901 (2006)) as a result of their activation by IL-4-producing CD4+ T cells (Martin, R. J. et al. J. Allergy Clin. Immunol. 119, 73-80 (2007)). Increased expression of BLT1 (leukotriene B4 receptor) on the surface of CD8+ T cells leads to their increased accumulation in the lungs (Guidelines for the diagnosis and management of asthma. Bethesda, Md.: National Institutes of Health National Heart Lung and Blood Institute; 2007). Both human (Miyahara, N. et al. J. Immunol. 172, 2549-2558 (2004)) and mouse (Miyahara, N. et al. J. Immunol. 174, 4979-4984 (2005)) CD8+ T cells demonstrate an insensitivity to corticosteroids not seen in CD4+ T cells, supporting the notion that CD8+ T cells are at the root of the failure of asthmatics to respond to corticosteroids and may be responsible for persistent AHR and inflammation (Koya, T. et al. J. Immunol. 179, 2787-2796 (2007)). In asthmatics, numbers of CD8+ T cells in the airways have correlated with lower airway function (LaVoie, H. A. and King, S. R. Exp. Biol. Med. 234, 880-907 (2009)).
Current therapies for allergic asthma have been fairly restricted with few new drugs introduced into the clinic in the last decade. Inhaled corticosteroids have remained the main anti-inflammatory agent for asthma. Indeed, upwards of 40-50% of asthmatics fail to respond to inhaled corticosteroids with changes in FEV1 (Hamelmann, E. et al. J. Exp. Med. 183, 1719-1729 (1996)). Moreover, corticosteroids may also enhance disease pathogenesis, especially amplifying responses in the steroid-insensitive population of CD8+ T cells (Miyahara, N. et al. Nature Med. 10, 865-869 (2004)). Corticosteroids may induce Th2 cytokine production while suppressing the production of Th1 cytokines. A combination of steroid insensitivity and plasticity of CD8+ T cells may be major contributors to the failure of some patients to respond to corticosteroids. CD8+BLT1+IL-13+ CD8+ T cells have been proposed to be a primary cause of the airway inflammation and hyperresponsiveness seen in asthma (National Asthma Education and Prevention Program (National Heart Lung and Blood Institute) Third Expert Panel on the Management of Asthma. National Center for Biotechnology Information (U.S.). Expert panel report 3 guidelines for the diagnosis and management of asthma. Bethesda, Md.: National Institutes of Health National Heart Lung and Blood Institute; 2007, Guidelines for the diagnosis and management of asthma. Bethesda, Md.: National Institutes of Health National Heart Lung and Blood Institute; 2007). However, the mechanism underlying the conversion of CD8+ T cells from IFN-γ-producing cells to IL-13 producing cells remains unclear.